DESCRIPTION: This is a proposal to analyze the correlations between chromatin structure, transcription rates, presence of DNA binding proteins and DNA methylation patterns with DNA damage, repair and mutagenesis in the p53 gene in cultured fibroblasts. Damage of DNA by environmental carcinogens is assumed to be a principal factor in the induction of mutations and the development of cancer in humans. The p53 tumor suppressor gene is the most frequently mutated gene in human malignancies. One popular hypothesis is that the spectrum of mutations found in different cancers may bear the signature of the mutagenesis process. To test this hypothesis Dr. Pfeifer will undertake a detailed analysis of the mutagenesis process at the p53 locus using in vitro systems. As a first step in this direction he will investigate in different cell types the chromatin structure at the critical p53 exons including an analysis of nucleosome association and complete DNA methylation patterns. To understand the mechanisms of mutagenesis, he will analyze DNA damage and DNA repair events at the DNA sequence level in human cells treated with several different mutagens and carcinogens. He will apply the ligation-mediated polymerase chain reaction (LMPCR), a uniquely sensitive technique for in vivo mapping of DNA adducts, to determine the frequency of a number of different mutagenic DNA adducts at each nucleotide position of the p53 gene's exons five through nine. Repair rates for some selected DNA adducts will be analyzed to determine if repair is sequence-specific. An increased adduct frequency and/or decreased repair rates at specific nucleotide positions may create mutation hot spots. The mutagenesis will be directly analyzed at selected p53 sites by using an in vitro mutation assay based on RFLP/PCR and single nucleotide primer extension. Methylation data, sequence-dependent adduc frequencies, repair rates, and data from the in vitro mutation assay will be compared with published frequencies of mutations found in a variety of human cancers including those for which involvement of an environmental carcinogen is suspected. Thus, he will attempt to provide in vitro model systems to study the basic molecular mechanisms that give rise to mutations in the p53 gene.